Laboratory of Microbial Ecology and Technology (LabMET)

1. Laboratory of Microbial Ecology and Technology (LabMET) Research topics and expertise Tom Van de Wiele, PhD

2. Mission Microbial Ecology Strategic research Applied research Technology

3. Strategic Research • Microbial communities • Quorum sensing • Electron shuttling • Horizontal gene transfer • Metabolomics

4. Applied Research • Environmental Microbiology • Wastewater • Microbial fuel cells • Biodegradation • Anaerobic treatment • Nitrogen removal strategies • Minimizing wast sludge

5. Applied Research • Environmental Microbiology • Soil / Sediments • Bioprecipitation of catalytic particles • Anaerobic removal of organochlorine contaminants • Soil and river sludge clean-up • Pesticide degradation and ecotoxicology

6. Applied Research • Environmental Microbiology • Solid Wastes • Solid waste treatment • De-icing • Buildings and structures • Biologically mediated CaCO3 formation • Microbial induced corrosion • Air • Indoor air pollution • Biotrickling filtration

7. Applied Research • Gastrointestinal Microbiology • Functional foods • Pro- / pre- / synbiotics • Bioactivation of food components • Rumen microbiology • Risk assessment • Environmental contaminants • Toxic food processing metabolites • Phage therapy

8. Applied Research • Foodchain Microbiology • Drinking water • Hygienisation • Water recycling • Pathogen abatement • Aquaculture systems • Habitat research • Epiphytes on grain • Space station life cycles • Deep sea methane oxidation

9. Tools and instruments • Reactor Technology • Microbial Fuel cells • Activated sludge systems • Upflow Anaerobic Sludge Bed reactors • Membrane reactors • Rotating disc reactors • Simulator of the Human Intestinal Microbial Ecosystem (SHIME) • Dialysis reactors

10. Tools and instruments • Molecular Analysis • PCR • DGGE • FISH • Realtime PCR • Cloning • Flow Cytometry

11. Tools and instruments • Microbial analysis • Epifluorescence and light microscopy • Growth kinetics • Microbial isolations and enrichments • Metabolic activity • Bioassays • Biodegradation assays

12. Tools and instruments • Physico – chemical analysis • Gas chromatography • HPLC • Ion chromatography • Spectrophotometry • Atom absorption • BOD, COD, TSS, VSS, NOX, TOC,…

13. Gastrointestinal microbial ecology Microbial biotransformation of environmental and food compounds in the gut and the consequences for biological activity assessment

14. Colonmicrobiota and health • Colon ascendens, colon transversum, colon descendens • Water- and salt resorption • Microbiota • 500 species, 1014 CFU/mL • ± stabile community

15. Colonmicrobiota and health Health effects: • Further digestion • Production of SCFA as energy source for colonocytes • Immunostimulation • Production of vitamins (K en B12) • Colonization resistance against pathogens • Formation of health-promoting components from food

16. Colon microbiota and health Health effects: • Colonization by pathogens • Formation of toxins • Putrefaction • Formation of (geno-)toxic compounds from food (contaminants) • Recent (!): microbiota stimulate fat uptake and synthesis

17. Colon microbiota and health A microbial community in balance (Gibson & Robertfroid ,1995)

18. SHIME-Tec: gastrointestinal in vitro technology Simulator of the Human Intestinal Microbial Ecosystem

19. Twin SHIME : parallel treatment and control

20. Oral exposure to bioactive compounds • Food: • Soy and hop isoflavones • Heterocyclic aromatic amines from grilled meat • ... • Environment: • Soil ingestion • Inhalation of dust and subsequent ingestion • ...

21. Case 1. Microbial conversion of phytoestrogens • Soy phytoestrogens: • Daidzine  daidzein  equol (microbial action) • Equol has beneficial health effects • Microbial consortium applicable as probiotic • K. Decroos et al. (2005) • Hop phytoestrogens: • Isoxanthohumol  hoppein (8-prenylnaringenin or 8-PN) • Carried out by colon microbiota • Importance for hop supplements, beer industry… • S. Possemiers et al. (2006)

22. Hop phytoestrogens • Beer: • Isoxanthohumol: mg/L range • 8-prenylnaringenin: traces • Menohop: food supplement for relief of menopausal symptoms • 100 g 8-PN / d (1 tablet / d) • IX: mg/L range

23. Large interindividual variability Batch incubation of IX with fecal microbiota from 51 women

24. 8-PN production in vitro • isoxanthohumol addition to SHIME • no conversion in ascending colon • conversion in transverse and especially descending colon • estrogenic activity as observed with estrogen bioassay • moderate in transverse colon • high in descending colon

25. In vitro - in vivo comparison • SHIME run with fecal microbiota from • woman A: high 8-PN producer • woman B: moderate 8-PN producer • woman C: low 8-PN producer • In vivo: urinary excretion of 8-PN corresponds to in vitro incubation

26. Biological activity assessment • Uptake of food supplement: • IX intake of 1 mg/d • 8-PN producing intestinal microbiota: • 8-PN exposure of more than 500 g/d • Range of biological activity • Risk assessment process needs to incorporate bioactivation by intestinal bacteria • Complexity: interindividual variability

27. Case 2. Oral exposure to PAHPolycyclic Aromatic Hydrocarbons • Ingestion of contaminated soil • Industrial and urban areas • Atmospheric deposition of PAH: 50 g.ha-1.yr-1 • Oral uptake • Adults: 50 mg.d-1 • Children: 200 mg.d-1 • Occasionally: 1-20 g.d-1 • HUMAN HEALTH RISK ASSESSMENT • Focus on intestinal absorption and bioactivation by human enzymes

28. Cytoplasm AhR Translate proteins Arnt mRNA DRE Nucleus Current knowledge on PAH bioactivation 3. Gene expression 1. PAH release from soil / nutrition 2. Intestinal absorption Intestine or liver cells 4. Possible bioactivation to toxic compounds

29. What happens to non-absorbed PAHs ? • Are colon microbiota capable of biotransforming PAHs? • Are microbial PAH metabolites bioactive?

30. Experimental set-up • Incubate PAH in samples from SHIME reactor • Screen for PAH metabolites • Estrogen receptor bioassay: estrogenicity • LC-ESI-MS: hydroxy-PAH • Pure PAH compounds • PAH contaminated soil samples

31. Yeast Estrogen test • Human estrogen receptor in yeast cell • Estrogen responsive elements in plasmid • Reporter gene lacZ

32. SHIME: colon microbiota activate PAHs

33. Chemical analysis • LC-ESI-MS: hydroxylation of PAHs • 1-OH pyrene: 4.3 µg/L • 7-OH B(a)P: 1.9 µg/L OH EE2 7-OH B(a)P

34. Urban playground soil sample: 50 ppm PAH

35. Biological activity assessment • PAH exposure • Adult: 5 g PAH/d Child:50 g PAH/d • Colon microbiota convert PAH to pseudo-estrogenic metabolites • Hydroxylation under anaerobic conditions? • Enterococcus faecalis • Mucosa associated bacteria: micro-aerophilic conditions • Relevant biological activity in vivo ?

36. Chemopreventive effect from prebiotics • Prebiotic inulin: add to SHIME reactor • Evaluate inulin as chemopreventive agent • Start-up, inulin treatment (2.5 g/d) • Incubate SHIME suspension with 40 µM B(a)P • Monitor PAH bioactivation with yeast estrogen bioassay • Relate to prebiotic effects • Metabolic analysis • PCR-DGGE-sequencing • Real-time PCR quantification Bifidobacterium sp.

37. Ascending colon: inhibitory effect

38. SCFA: colon ascendens • 26% increase ** • Towards propionic and butyric acid • Reversible effect

39. Case-study: inuline (Resultaten) • PCR-DGGE van bifidobacteria 3 1 2 1. Bifidobacterium sp. 2. Bifidobacterium infantis (96% sim.) Inulin treatment samples 3. Bifidobacterium longum (95% sim.) Start-up and control samples INULINE: stimulatie van de BIFIDOBACTERIA

40. Take home messages • Metabolic potency from gut microbiota • Higher than currently anticipated • Consider this process for risk assessment • Interindividual variability ! • Identification of responsible bacteria and process conditions needed • Modulation of biological activation through dietary factors, microbial community composition...

41. Contact information LabMET – Ghent University Coupure Links 653 B-9000 Gent http://labMET.ugent.be/ http://www.shimetec.be +32/9/264.59.76